Selenoproteins are proteins in which the amino acid, selenocysteine, is incorporated cotranslationally at UGA codons. Studies of selenoproteins over the last decade or so have focused on two major areas, identification of new selenoproteins and elucidation of their functions, and unraveling the mechanism by which the cellular translational machinery directs incorporation of this amino acid at a "stop" codon. The signals in mRNAs conferring selenocysteine incorporation, termed SECIS elements, were first identified by the investigator during her postdoctoral studies. Recently, two factors that mediate selenocysteine incorporation were identified, EFsec and SBP2. Our laboratory identified and is characterizing the selenocysteyl-tRNA specific elongation factor, EFsec, which mediates incorporation of this unusual amino acid, through interaction with the SECIS binding protein, SBP2. We have also demonstrated interaction of EFsec with SBP2, and the role of selenocysteyl-tRNA in mediating this interaction. Other factors have been implicated in this process, including two of unknown function, SECp43 and SLA/LP, and two of known function, selenophosphate synthase and selenocysteine lyase. We are expressing and characterizing these proteins with the goal of understanding how they function to mediate selenocysteine biosynthesis and incorporation into proteins. Specific aims to be addressed are: I. Investigate functions of SECp43, SLA/LP and selenocysteine lyase in selenoprotein synthesis. II. Investigate subcellular localization and nucleocytoplasmic transport of EFsec and SBP2, and the implications for nonsense mediated decay and the hierarchy of selenoprotein synthesis. III. Investigate interactions between EFsec, SBP2 and their interaction partners, and the functions of these interaction partners in selenoprotein synthesis.